The overall objective of the proposed project is to gain novel information regarding how components of particulate matter (PM) air pollution and microbial stimuli synergistically interact to promote inflammatory-like processes in the lung, and thereby contribute to the onset of respiratory disease. Numerous epidemiological studies have linked exposure to PM with chronic respiratory and cardiovascular diseases. Studies have shown nickel, a common component of PM, mediates some of the biological effects of PM both in vitro and in vivo. Chronic inflammatory diseases are also associated with colonization by infectious agents, including Mycoplasma fermentans. One of the hallmarks of inflammation is increased elaboration of prostaglandins (PG) through the induction of the cyclooxygenase-2 enzyme (prostaglandin-endoperoxidase synthase 2, PTGS2). Preliminary data show that exposure to NiS04 and the M. fermentans-derived lipopeptide MALP-2 synergistically enhances expression of PTGS2 and the immune-modulating chemokine CXCL8(IL-8) in cultured human lung fibroblast cells (HLF). Concurrent with the induction of PTGS2 and CXCL8, Ni and MALP-2 synergistically stimulate PGE2 and CXCL8 protein release from HLF. The proposed study will explore the hypothesis that PTGS2-derived PGE2 contributes to the amplification of CXCL8 production from HLF during mixed exposures to Ni and microbial stimuli. The specfic aims are to 1) determine the cellular and molecular mechanisms underlying the synergistic induction of PTGS2 expression in HLF in response to concurrent Ni and MALP-2 exposure and 2) determine the mechanistic relationship between the production of PGE2 following mixed exposures to Ni and MALP-2 and subsequent production of CXCL8. Luciferase reporter plasmids driven by human PTGS2 and CXCL8 promoters along with electrophoretic mobility shift assays (EMSA) will be employed to determine how Ni and MALP-2 synergistically enhance activation of PTGS2 and CXCL8. Relevant cis-regulatory DNA binding elements and transactivating protein factors responsible will be mapped using full-length, truncated and mutant versions of the PTGS2 and CXCL8 promoters. siRNA along with pharmacological agonists and antagonists will be used to probe the specific contribution of PTGS2, PGE2 and PGE2 receptor subtypes involved in transducing Ni and MALP-2-induced CXCL8 expression and subsequent protein release from HLF. Real-life environmental exposures are likely to involve a mixture of toxicants and microbial stimuli. Results from this study will increase our understanding of how metals such as Ni can sensitize cells to microbial-driven inflammation in the lung, and how toxicant and microbial stimuli interact to promote the onset of respiratory disease in human populations. [unreadable] [unreadable] [unreadable]